Recent work from this laboratory has demonstrated that the synthesis of mRNAs for the ribosomal proteins of Saccharomyces cerevisiae are coordinately regulated under a variety of experimental conditions in spite of the fact that the genes for ribosomal proteins appear to be widely scattered on the genome. This application attempts to understand this phenomenon using a combined biochemical and genetic approach. Yeast DNA will be "shotgunned " into lambda bacteriophage generally according to the methods of Davis (Cell 8, 227, 1976). Hybrid phage carrying yeast genes that are transcribed with some frequency will be identified by hybridization with radioactive mRNA or its cDNA. We have identified yeast ts mutants which are severely deficient in mRNA for ribosomal proteins at the restrictive temperature. By comparing the hybridization of mRNA from cells cultured at the permissive and restrictive temperatures with lambda hybrids, we expect to identify phage which appear to carry ribosomal protein genes. To verify that any one hybrid indeed carries a ribosomal protein gene, we will measure the effect of its DNA on the synthesis of ribosomal proteins in vitro by several techniques. Once hybrids carrying a ribosomal protein gene are identified, we will ask (a) what is the size of the transcript for the mRNA for a ribosomal protein? (b) Is there any clustering of riobosomal protein genes? (c) By using further restriction enzyme analysis, can we isolate a fragment of DNA which contains the ribosomal protein gene and its regulatory region and not much else? (d) Eventually, by sequencing the regions adjacent to the 5' end of the mRNA for several ribosomal protein genes, can we identify a sequence which is responsible for coordinate transcription of the genes? Concurrently, we will be carrying out a genetic approach to identify the location of ribosomal protein genes and to isolate mutants in them. Disomic strains will be used to identify ribosomal protein genes by a gene dosage effect. Unselected temperature sensitive mutations will be screened for changes in ribosomal proteins by 2D electrophoresis, since this has proved to be an effective technique in E. coli.